Collagen is one of the most predominant components of the extracellular matrix, and is the main component of fascia, cartilage, ligaments, tendons, bone and skin. Along with soft keratin, it is responsible for skin strength and elasticity, it strengthens blood vessels and plays a role in tissue development. It is also present in crystalline form in the cornea and lens of the eye. The structural and mechanical properties of collagen arise from property of collagen peptides to form tough bundles called collagen fibers. Tropocollagen is a triple helix formed by collagen peptides, and is a subunit of larger collagen aggregates such as fibrils, which in turn form even larger aggregates such as fibers. Tropocollagen is approximately 300 nm long and 1.5 nm in diameter, and made up of three polypeptide strands, each possessing the conformation of a left-handed helix. Tropocollagen peptides each include a high content of three repeating Xaa-Yaa-Gly strands, where Pro-Hyp-Gly (Hyp=(2S,4R)-4-Hydroxyproline) is one of the more abundant repeating unit. These three left-handed helices are twisted together into a right-handed triple helix coiled coil, a quaternary structure stabilized by numerous hydrogen bonds. The tropocollagen subunits spontaneously self-assemble, with regularly staggered ends, into even larger arrays in the extracellular spaces of tissues. In the fibrillar collagens, the molecules are staggered from each other by about 67 nm, which is variable depending upon the degree of hydration. The triple-helices are also arranged in a hexagonal or quasi-hexagonal array in cross-section, in both the gap and overlap regions.
Twenty-nine types of collagen have currently been identified, though over 90% of the collagen in the body, however, are of type I, II, III, and IV, with type I being the most common. Type I collagen is found in skin, tendon, vascular, ligature, organs, and is the main component of bone; type II collagen is the main component of cartilage; type III collagen is the main component of reticular fibers, and is commonly found alongside type I; and type IV collagen forms the bases of cell basement membrane.
Though many tissues are composed primarily of type I collagen including tendon, ligament, skin, and bone, each of these structures also contains other collagen types, and also proteoglycans and glycosaminoglycans, and minerals in the case of bone. The dramatic difference in mechanical properties of each of these collagen structures has been reported to be primarily due to the molecular and macromolecular organization of collagen and the interplay of those substructures with other non-collagen type I components.
Though nearly 30 different types of natural collagen have been identified, the tertiary structures of each share the motif of the collagen triple helix. The collagen triple helix (CTH) motif is composed of three chains that each adopt a left-handed helix, which consists of 3 residues/turn (Bella et al., Science, 266:75-81 (1994); Cowan et al., Nature, 176:1062-4 (1955)). These chains come together to form a right-handed superhelix. A distinctive feature of collagen is the regular arrangement of amino acids in each of the three chains of these collagen subunits. The sequence often follows the pattern Xaa-Yaa-Gly, Gly-Pro-Xaa and/or Gly-Xaa-Hyp, where Xaa and Yaa may be any of various other amino acid residues. Proline or hydroxyproline constitute about ⅙ of the total sequence. There is some covalent crosslinking within the triple helices, and a variable amount of covalent crosslinking between tropocollagen helices forming well organized aggregates, called fibrils. Larger fibrillar bundles, or fibers, are formed from aggregates of fibrils and with the aid of and inclusion of other different classes of proteins, such as glycoproteins and proteoglycans. In addition, in certain specialized tissues like bone, the collagen triple helices lie in a parallel, staggered array with gaps of about 40 nm between the ends of the tropocollagen subunits. It has been reported that such gaps may serve as nucleation sites for the deposition of hydroxyapatite and other mineral components in long, hard, fine crystals. Type I collagen has also been reported to be largely responsible for the high tensile strength of bone.
Minimal peptide sequences based on the idealized proline-hydroxyproline-glycine (POG) sequence have been found to adopt the CTH motif (Persikov et al., Biochemistry, 39:14960-7 (2000)). Goodman and coworkers have found that six repeating units of POG are the minimum length required for triple helix formation at room temperature (Kwak et al., Bioorg Med Chem, 7:153-60 (1999)). Furthermore, increasing the number of repeating units was found to increase the triple helix stability.
Collagen has the ability to self-associate in vitro, forming gels that can act as a 3-dimensional substrate, and provide mechanical and biological signals for cell growth. Research on collagen fibrillogenesis with and without additional extracellular matrix components has been directed to a better understanding of the interrelationship between collagen and other extracellular matrix molecules in tissues. However, natural collagen matrices, such as MATRIGEL, are inherently heterogeneous materials that routinely vary in composition, thus complicating the analysis of bioassays. Furthermore, those matrices do not easily allow for the precise introduction of biomolecules, such as cell adhesion agents and growth factors. (see, e.g., Alavi et al., 426:85-101 (2007); Horch et al., J. Cell Mol. Med., 9:592-608 (2005); Ramachandran et al., Biodrugs, 20:263-69 (2006)). The use of natural collagen for tissue engineering is limited due to the difficulty in the precise control of scaffold morphology and limited ability to introduce chemical diversity.
Thus, of interest are synthetic collagen fibers that can be used not only to mimic native collagen but also to enhance its biological roles (Koide, T., Connect. Tissue Res., 46:131-41 (2005)). Self-assembling synthetic peptides have been explored as an alternative source of collagen material in an attempt to mimic and expand on the properties associated with collagen. Several self-assembling collagen mimetic peptides have been described (Paramonov et al., Macromolecules, 38:7555-7561 (2005); Kotch et al., Proc. Natl. Acad. Sci. USA, 103:3028-3033 (2006); Rele et al., J. Am. Chem. Soc., 129:14780-14787 (2007); Cejas et al., Proc. Natl. Acad. Sci. USA, 105:8513-8518 (2008); Przybyla et al., J. Am. Chem. Soc., 130:12610-12611 (2008); Pires et al., J. Am. Chem. Soc., 131:2706-2712 (2009)). To date, however, these collagen based self-assembling systems have not been exploited as 3-dimensional scaffolds for cell encapsulation and cell culture.
Another approach is to create synthetic collagen fibers that can be used to mimic native collagen and also to enhance its biological roles by generating small collagen peptides that self-assemble into collagen fibers. However, only self-assembling collagen fibers employing linear growth through incorporation of a variety of N and C-terminal functional groups has been reported. Specifically, electrostatic interactions (Koide, T., Connect. Tissue Res., 46:131-41 (2005)), π-π stacking (Koide, T., Connect. Tissue Res., 46:131-41 (2005); Cejas et al., J. Am. Chem. Soc., 129:2202-3 (2007)), a modified cysteine knot (Kotch et al., Proc. Natl. Acad. Sci. USA, 103:3028-3033 (2006)), and native chemical ligation (Paramonov et al., Macromolecules, 38:7555-7561 (2005)) have been implemented.
In addition, scaffolds composed of either non-bioactive polymers or naturally derived biopolymers have been reported (Fischbach et al., Proc. Natl. Acad. Sci. U.S.A., 106:399-404 (2009); Griffith & Swartz, Nature Rev. Mol. Cell. Biol., 7:211-224 (2006); Lavik et al., Biomaterials, 26:3187-3196 (2005)). Covalently cross linked polymers based on polyethylene oxide (PEO), poly(L-lactide) (PLLA), and poly(lactide-co-glycolic acid) (PLGA) have also been reported (Langer et al., Vacanti, Science, 260:920-926 (1993); Anderson et al., Adv. Drug. Deliv. Rev., 28:5-24 (1997); Langer et al., Nature, 428:487-492 (2004); Kong et al., Nat. Rev. Drug Discovery, 6:455-463 (2007)). Further avenues include peptide-based materials that mimic aspects of the 3-dimensional matrix of cells, such as self-assembling peptide amphiphiles (Hartgerink et al., Science, 294:1684-1688 (2001)), α-helices (Ryadnov et al., Nat. Mater., 2:329-332 (2003); Zhou et al., J. Am. Chem. Soc., 126:734-735 (2004); Lazar et al., Biochemistry, 44:12681-12689 (2005); Zimenkov et al., J. Am. Chem. Soc., 128:6770-6771 (2006); Dong et al., J. Am. Chem. Soc., 130:13691-13695 (2008)), β-sheets (Zhang et al., Proc. Natl. Acad. Sci. USA, 90:3334-3338 (1993); Haines-Butterick et al., Proc. Natl. Acad. Sci. USA, 104:7791-7796 (2007); Murasato et al., Biomacromolecules, 9:913-918 (2008)), and β-amino acid helices (Pomerantz et al, Angew. Chem. Int. Ed., 47:1241-1244 (2008); Angew. Chem., 47:1241-1244 (2008)).
While these strategies have been successful in generating collagen peptide fibers, there is still a need to control the three-dimensional architecture of collagen networks, a desirable feature for tissue engineering.
Described herein are synthetic collagen conjugates. In one aspect, the conjugates are capable of spontaneous self-assembly, or self-assembly under mild conditions, into triple helical configurations, also referred to as CTHs. It is appreciated that such triple helical configurations or CTHs are analogous to tropocollagen. In another aspect, the CTHs formed from the conjugates described herein are also capable of aggregating in the presence of metal ions to form fibrils, fibers, and/or more complex bundled structures. In another embodiment, the synthetic collagen conjugates described herein for 3-dimensional structures or aggregates with myriad morphologies, including particle morphologies, mesh morphologies, or mesh morphologies with embedded particulate regions. Illustrative particle morphologies include, spheres, nanospheres, hollow microspheres, open curved tubes, layered sheets, C-types, microdisks, nanodisks, shaped flakes, florettes, cages, meshes, anemones, microflorettes, and the like. Illustrative mesh morphologies include a wide range of porosity. Each of those aggregates may have nanometer and/or micrometer scale features. It has been discovered herein that the morphologies of the aggregates are controlled, at least in part, by the nature of the metal, the nature of the synthetic collagen conjugates, and the relative concentration of the metal.
Accordingly, described herein are synthetic collagen conjugate aggregates with ordered structures and with tunable shapes, sizes, and tertiary structure. Without being bound by theory, it is believed herein that the core elements of the aggregates should be composed of short, readily synthesized monomers that are capable of self-assembling either spontaneously or under mild conditions, and are also capable of forming larger aggregates under the influence of an external stimulus, such as in the presence of a metal ion. In addition, described herein are synthetic collagen conjugate aggregates with tunable physical properties, such as mechanical strength, tensile strength, porosity, and the like.
In another embodiment, the synthetic collagen conjugates described herein are covalent conjugates of one or more metal binding moieties and a peptide. It is understood that the any or all of the metal-binding moieties may be directly attached to the peptide, or optionally covalently attached to the peptide through a divalent linker. In one aspect, the peptide is formed from and analogous to collagen-like material. It is appreciated that such peptides may possess similar physical and biomechanical properties to natural collagen, thereby generating a scaffold that may more closely mimic the extracellular matrix (ECM). In another aspect, the conjugate includes one or more metal-binding moieties, and a peptide comprising tripeptides of glycine, tripeptides of glycine and proline, tripeptides of glycine and hydroxyproline, and tripeptides of glycine, proline and hydroxyproline. In another aspect, the one or more metal-binding moieties are covalently attached to the peptide. In another aspect, the synthetic collagen conjugates described herein are capable of forming type II helix. Without being bound by theory, it is believed herein that three-dimensional, collagen-peptide assemblies could be obtained by the appropriate positioning of metal-binding ligands within collagen triple helices, with the underlying design criteria being to incorporate metal binding sites into small collagen peptides and use metal-ligand interactions to drive aggregation. It is appreciated however that the modified peptides should be able self assemble or assemble under mild conditions into triple helices.
Several non-limiting illustrative designs are described herein (see, FIG. 1, illustrated with a polyPOG peptide core). In one embodiment, one or more metal binding moieties are incorporated at both of the termini of one or more of the peptides forming the CTH (FIG. 1A, linear aggregation). In one variation, one or more metal binding moieties are incorporated within the interior of the one or more of the peptides forming the CTH (FIG. 1A, radial aggregation). In another variation, one or more metal binding moieties are incorporated within the interior, and at either one of or, alternatively, at both of the termini of one or more of the peptides forming the CTH (FIG. 1A, crosslinked aggregation). In another embodiment synthetic collagen conjugates are described that are capable of aggregating in a linear manner. In another embodiment synthetic collagen conjugates are described that are capable of aggregating in a radial manner. In another embodiment synthetic collagen conjugates are described that are capable of aggregating in both a linear and a radial manner, resulting in a cross-linked aggregate.
Also described herein are uses for synthetic collagen conjugates in 3-dimensional cell culture, for cell adhesion, in tissue engineering and regeneration, in cosmetic surgery, in the construction of artificial skin substitutes, in the management of severe burns and burns surgery, in reconstruction of bone and in a wide variety of dental, orthopedic and surgical purposes. Also described herein are uses for synthetic collagen conjugates as drug delivery vehicles. Also described herein are uses for synthetic collagen conjugates for delivering populations of cells to a site of disease or injury. Also described herein are methods for treating diseases and/or injuries that include administration of the compounds and compositions, and/or the resulting aggregated prepared therefrom, described herein to a patient in need of relief from the disease or injury. In one embodiment, the methods are used for directing cell adhesiveness. In another embodiment, methods are used to deliver populations of cells. It is understood that the aggregates described herein are advantageously compatible with living cells.
In another embodiment, the aggregates described herein are reversible and may be converted back to smaller subunits such as triple helical structures. It is appreciated that such reversibility may be advantageous to allow for the release of drugs and/or cells from the interior of the aggregate structure after administration. It is further appreciated that such a reversible property is precluded in most covalently cross-linked polymers.
In another embodiment, synthetic collagen conjugate aggregates are described herein where one or more un-coordinated, uncomplexed, or unbound metal binding moiety is used to deliver a drug. The unbound metal binding moiety may be used for binding and temporal release of biologically relevant molecules, such as growth factors, associated with the unbound metal binding moiety. It is understood that the unbound metal binding moiety may be on the surface or in the interior of the aggregate.
As used herein, associated refers to molecules that are covalently attached, complexed, ionically bonded, attached via a conjugate such as avidin-streptavidin, biotin-streptavidin, and the like.
In another embodiment, synthetic collagen conjugate aggregates are described herein and used to stabilize and/or deliver a cell or population of cells. In another embodiment, synthetic collagen conjugate aggregates are described herein where one or more un-coordinated, uncomplexed, or unbound metal binding moiety is used to stabilize and/or deliver a cell or population of cells. The unbound metal binding moiety may be used for binding and temporal release of a cell using a cell adhesion agent or peptide associated with the unbound metal binding moiety. In yet another embodiment, it was envisioned that collagen peptide biomaterials may be used as delivery vehicles for in vivo cell-based therapies with regenerative applications. It is understood that the unbound metal binding moiety may be on the surface or in the interior of the aggregate. In each of the embodiments described herein, cells may be a population of exogenously grown cells.
In another embodiment, synthetic collagen conjugate aggregates are described herein where one or more un-coordinated, uncomplexed, or unbound metal binding moiety is used as a scaffold and/or vehicle for stem cell differentiation and cell growth into tissue. The unbound metal binding moiety may be used for binding and temporal release of a cell using a cell adhesion agent or peptide associated with the unbound metal binding moiety. It is understood that the unbound metal binding moiety may be on the surface or in the interior of the aggregate. Without being bound by theory, it is believed herein that the physical properties of synthetic collagen peptide aggregates play a role in cell growth and differentiation, and that these properties may be modified in a predetermined way as described herein.
In another embodiment, methods are described for promoting tissue regeneration using synthetic collagen conjugate aggregates. In another embodiment, methods are described for promoting the growth and differentiation of stem cells, including adult stem cell, using synthetic collagen conjugate aggregates. In another embodiment, methods are described for promoting the growth of blood vessels using synthetic collagen conjugate aggregates. In one aspect, the methods comprise the step of administering one or more synthetic collagens, either alone or in combination with other components to the patient, where the one or more synthetic collagens promote healing, tissue regeneration, or prevent injury of the tissue in the patient. The methods and compositions described herein can be used to treat any condition where the tissue is damaged, including damaged connective tissue, such as cartilage, muscle tissue, and bone tissue.